The identification of Tautoneura mori as the vector of mulberry crinkle leaf virus and the infectivity of infectious clones in mulberry

2021 ◽  
Author(s):  
Lu Quanyou ◽  
Yu Ma ◽  
William Kojo Smith ◽  
Jing Yu ◽  
Yong Yuan Cheng ◽  
...  
Keyword(s):  
Binary Vector ◽  
Post Inoculation ◽  
Infectious Clones ◽  
Natural Vector ◽  
First Time ◽  
Leaf Virus ◽  
Inoculation Assay

Mulberry crinkle leaf virus (MCLV) is a novel geminivirus identified from mulberry. The pathogenicity and the natural vector of transmission have remained unknown for MCLV. Here, the infectious clones which consisted of the complete tandem dimeric genome of MCLV in a binary vector were constructed and agro-inoculated into mulberry seedlings. The results showed that the infectious clones of MCLV were systemically infectious to mulberry, but the infected mulberry plants did not show any virus-like symptoms. The natural transmission vectors of MCLV were also identified from possible vector insects occurring on the MCLV-infected mulberry plants. The vector ability of Tautoneura mori Matsumura was identified through inoculation assay. Three of 21 (14.3%) seedlings inoculated with T. mori collected from MCLV-infected mulberry plants grown naturally were detected to be MCLV-positive 50 days post-inoculation. These MCLV-positive mulberry plants did also not show any virus-like symptoms. Collectively, it is suggested that MCLV is infectious to mulberry plants, but MCLV alone does not induce symptoms. The leafhopper T. mori was for the first time determined experimentally to be a transmission vector of MCLV.

scholarly journals Tomato Twisted Leaf Virus: A Novel Indigenous New World Monopartite Begomovirus Infecting Tomato in Venezuela

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 327 ◽  
Author(s):  
Gustavo Romay ◽  
Francis Geraud-Pouey ◽  
Dorys Chirinos ◽  
Mathieu Mahillon ◽  
Annika Gillis ◽  
...  
Keyword(s):  
Crop Production ◽  
New World ◽  
Systemic Infection ◽  
Plant Viruses ◽  
Monopartite Begomoviruses ◽  
Bipartite Begomoviruses ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Infectious Clones ◽  
Leaf Virus

Begomoviruses are one of the major groups of plant viruses with an important economic impact on crop production in tropical and subtropical regions. The global spread of its polyphagous vector, the whitefly Bemisia tabaci, has contributed to the emergence and diversification of species within this genus. In this study, we found a putative novel begomovirus infecting tomato plants in Venezuela without a cognate DNA-B component. This begomovirus was genetically characterized and compared with related species. Furthermore, its infectivity was demonstrated by agroinoculation of infectious clones in tomato (Solanum lycopersicum) and Nicotiana benthamiana plants. The name Tomato twisted leaf virus (ToTLV) is proposed. ToTLV showed the typical genome organization of the DNA-A component of New World bipartite begomoviruses. However, the single DNA component of ToTLV was able to develop systemic infection in tomato and N. benthamiana plants, suggesting a monopartite nature of its genome. Interestingly, an additional open reading frame ORF was observed in ToTLV encompassing the intergenic region and the coat protein gene, which is not present in other closely related begomoviruses. A putative transcript from this region was amplified by strand-specific reverse transcription-PCR. Along with recent studies, our results showed that the diversity of monopartite begomoviruses from the New World is greater than previously thought.

scholarly journals First Detection and Molecular Characterization of Apple Stem Grooving Virus, Apple Chlorotic Leaf Spot Virus, and Apple Hammerhead Viroid in Loquat in Spain

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2293
Author(s):  
Celia Canales ◽  
Félix Morán ◽  
Antonio Olmos ◽  
Ana Belén Ruiz-García
Keyword(s):  
Leaf Spot ◽  
High Throughput Sequencing ◽  
Eriobotrya Japonica ◽  
Spot Virus ◽  
Apple Stem Grooving Virus ◽  
Apple Stem ◽  
Chlorotic Leaf ◽  
First Time ◽  
Leaf Virus

Loquat (Eriobotrya japonica) is an important crop in Spain. To date, only one viral species, apple stem pitting virus (ASPV), has been detected in Spanish loquat orchards. In this study, the presence of additional viruses infecting this crop in Spain was investigated. RT-PCR and high-throughput sequencing (HTS) of symptomatic loquat plants led to first-time detection and characterization of apple stem grooving virus (ASGV), also known as citrus tatter leaf virus (CTLV), and apple chlorotic leaf spot virus (ACLSV) from Spain with description of nearly complete genomic sequences. The frequency of ACLSV infection was the highest, with over 30% of the samples testing positive and were also detected as coinfections with ASGV and ASPV, although most of the samples infected were symptomless. Studies on all the full-length sequences available in the databases were performed in order to establish the phylogenetic relationships of the Spanish isolates of these two viral species. Moreover, apple hammerhead viroid (AHVd) was also detected to infect loquat, the first host different from apple reported for this viroid to date.

scholarly journals Reconstruction and Characterization of Full-Length Begomovirus and Alphasatellite Genomes Infecting Pepper through Metagenomics

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 202 ◽  
Author(s):  
Verónica A. Bornancini ◽  
José M. Irazoqui ◽  
Ceferino R. Flores ◽  
Carlos G. Vaghi Medina ◽  
Ariel F. Amadio ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Full Length ◽  
Yellow Spot ◽  
Streak Virus ◽  
Rolling Circle ◽  
Viral Particles ◽  
Bipartite Genome ◽  
First Time ◽  
Leaf Virus

In northwestern Argentina (NWA), pepper crops are threatened by the emergence of begomoviruses due to the spread of its vector, Bemisia tabaci (Gennadius). The genus Begomovirus includes pathogens that can have a monopartite or bipartite genome and are occasionally associated with sub-viral particles called satellites. This study characterized the diversity of begomovirus and alphasatellite species infecting pepper in NWA using a metagenomic approach. Using RCA-NGS (rolling circle amplification-next generation sequencing), 19 full-length begomovirus genomes (DNA-A and DNA-B) and one alphasatellite were assembled. This ecogenomic approach revealed six begomoviruses in single infections: soybean blistering mosaic virus (SbBMV), tomato yellow spot virus (ToYSV), tomato yellow vein streak virus (ToYVSV), tomato dwarf leaf virus (ToDfLV), sida golden mosaic Brazil virus (SiGMBRV), and a new proposed species, named pepper blistering leaf virus (PepBLV). SbBMV was the most frequently detected species, followed by ToYSV. Moreover, a new alphasatellite associated with ToYSV, named tomato yellow spot alphasatellite 2 (ToYSA-2), was reported for the first time in Argentina. For the Americas, this was the first report of an alphasatellite found in a crop (pepper) and in a weed (Leonurus japonicus). We also detected intra-species and inter-species recombination.

scholarly journals First Report of Cherry necrotic rusty mottle virus Infecting Sweet Cherry Trees in Korea

Plant Disease ◽  
2014 ◽  
Vol 98 (1) ◽  
pp. 164-164 ◽  
Author(s):  
I. S. Cho ◽  
G. S. Choi ◽  
S. K. Choi ◽  
E. Y. Seo ◽  
H. S. Lim
Keyword(s):  
Sweet Cherry ◽  
Mottle Virus ◽  
Post Inoculation ◽  
Rt Pcr ◽  
First Report ◽  
Coat Protein Region ◽  
Pcr Products ◽  
Leafroll Virus ◽  
Leaf Virus ◽  
Cherry Trees

Cherry necrotic rusty mottle virus (CNRMV), an unassigned member in the family Betaflexiviridae, has been reported in sweet cherry in North America, Europe, New Zealand, Japan, China, and Chile. The virus causes brown, angular necrotic spots, shot holes on the leaves, gum blisters, and necrosis of the bark in several cultivars (1). During the 2012 growing season, 154 sweet cherry trees were tested for the presence of CNRMV by RT-PCR. Samples were randomly collected from 11 orchards located in Gyeonggi and Gyeongsang provinces in Korea. RNA was extracted from leaves using the NucliSENS easyMAG system (bioMérieux, Boxtel, The Netherlands). The primer pair CGRMV1/2 (2) was used to amplify the coat protein region of CNRMV. Although none of the collected samples showed any notable symptoms, CNRMV PCR products of the expected size (949 bp) were obtained from three sweet cherry samples from one orchard in Gyeonggi province. The PCR products were cloned into a pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of the three Korean sequences obtained (GenBank Accession Nos. AB822635, AB822636, and AB822637) showed 97% nucleotide sequence identity with a flowering cherry isolate from Japan (EU188439), and shared 98.8 to 99.6% nucleotide and 99.6 to 100% amino acid similarities to each other. The CNRMV positive samples were also tested for Apple chlorotic leaf spot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry rasp leaf virus (CRLV), Cherry leafroll virus (CLRV), Cherry virus A (CVA), Little cherry virus 1 (LChV-1), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV) by RT-PCR. One of the three CNRMV-positive samples was also infected with CVA. To confirm CNRMV infection by wood indexing, Prunus serrulata cv. Kwanzan plants were graft-inoculated with chip buds from the CNRMV-positive sweet cherry trees. At 3 to 4 weeks post-inoculation, the Kwanzan plants showed quick decline with leaves wilting and dying; CNRMV infection of the indicators was confirmed by RT-PCR. To our knowledge, this is the first report of CNRMV infection of sweet cherry trees in Korea. Screening for CNRMV in propagation nurseries should minimize spread of this virus within Korea. References: (1) R. Li and R. Mock. Arch. Virol. 153:973, 2008. (2) R. Li and R. Mock. J. Virol. Methods 129:162, 2005.

scholarly journals Symptom Severity of Nicotiana benthamiana Plants Inoculated with Agrobacterium Containing Infectious DNA-A Clones of Honeysuckle Yellow Vein Virus (HYVV)

2019 ◽  
Vol 7 (1) ◽  
pp. 12-20
Author(s):  
Sung Oh ◽  
Chang Won Choi
Keyword(s):  
Symptom Severity ◽  
Nicotiana Benthamiana ◽  
Virulence Gene ◽  
Peak Level ◽  
Yellow Vein ◽  
Post Inoculation ◽  
Standard Curve ◽  
Infectious Clones ◽  
A Genome ◽  
Young Leaves

To investigate the pathogenicity and virulence of the Honeysuckle yellow vein virus (HYVV) lacking betasatellites, PCR amplified unit-lengths of DNA-A genome of HYVV-[DJ] were cloned into binary vector pRI101-AN, and generated HYVV-[DJ]-1mer, -1.3mer and -2mer genomes. Each construct was transformed into Agrobacterium cells and agro-inoculated into young leaves of Nicotiana benthamiana. Except for the HYVV-[DJ]-1mer, HYVV-[DJ]-1.3mer and -2mer clones caused pronounced disease symptoms in N. benthamiana. HYVV-[DJ]-2mer agro-inoculated plants showed more severe plant stunting with downward leaf curling and crinkling than those of HYVV-[DJ]-1.3mer agro-inoculated plants. To discriminate the clone’s virulence quantitatively, SYBR Green-based real-time PCR was performed for the quantification of the target virulence gene DNA in agro-inoculated plants that were collected at weekly intervals for 4 weeks. Regression analysis was obtained from the standard curves by plotting Ct values over the logarithm of the amount of V1 protein gene DNA present in a dilution series of plasmid containing the full-length HYVV-[DJ] genome. Equation of the HYVV V1 DNA standard curve was used to quantify V1 gene DNA concentration in agro-inoculated plants with each clone. The accumulation of V1 gene DNA in HYVV-[DJ]-1.3mer agro-inoculated plants reached the peak level at 4 weeks post inoculation, while the accumulation of V1 gene DNA in HYVV-[DJ]-2mer agro-inoculated plants reached the peak level at 3 weeks post inoculation. The amount of V1 DNA in HYVV-[DJ]-1.3mer agro-inoculated plants was significantly more than that in HYVV-[DJ]-2mer agro-inoculated plants. Considering the results, there was a difference between the accumulation of virus DNA and the symptom severity of the analyzed plants agro-inoculated with each clone. It suggested that the infectious clones’ virulence is not necessarily correlated with the symptom severity.

The fine structure of sexual stage development and sporogony ofCryptosporidium parvumin cell-free culture

Parasitology ◽  
2016 ◽  
Vol 143 (6) ◽  
pp. 749-761 ◽  
Author(s):  
HEBATALLA M. ALDEYARBI ◽  
PANAGIOTIS KARANIS
Keyword(s):  
Cryptosporidium Parvum ◽  
Culture System ◽  
Post Inoculation ◽  
Large Area ◽  
Large Nucleus ◽  
Transmission Electron ◽  
A Cell ◽  
Stage Development ◽  
Sexual Stages ◽  
First Time

SUMMARYThe sexual stages and new oocysts development ofCryptosporidium parvumwere investigated in a cell-free culture system using transmission electron microscopy (TEM). Sexual development was extremely rapid after inoculation of oocysts into the medium. The process began within 1/2–12 h and was completed with new oocyst formation 120 h post-inoculation. The macrogamonts were bounded by two membranes and had amylopectin granules and two distinct types of wall-forming bodies. The microgamonts had a large nucleus showing lobe projections and condensation of chromatin, giving rise to peripherally budding microgametes. The microgametes contained a large area of granular substance containing groups of microtubules surrounding the electron-dense nucleus. In some instances, the dividing microgamy was observed in cell-free cultures with no preceding merogonic process. Fertilization was observed with the bullet-shaped microgamete penetrating an immature macrogamont at 24 and 216 h. The new thin- and thick-walled oocysts had a large residuum with polysaccharide granules and sporogony noted inside these oocysts. Novel immature four-layer walled thick oocysts with irregular knob-like protrusions on the outer layer resembling the immatureEimeriaoocysts were also observed. The present study confirms the gametogony and sporogony ofC. parvumin cell-free culture and describes their ultra-structure for the first time.

scholarly journals Scale Drop Disease Virus Associated Yellowfin Seabream (Acanthopagrus latus) Ascites Diseases, Zhuhai, Guangdong, Southern China: The First Description

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1617
Author(s):  
Yuting Fu ◽  
Yong Li ◽  
Weixuan Fu ◽  
Huibing Su ◽  
Long Zhang ◽  
...  
Keyword(s):  
Disease Virus ◽  
Southern China ◽  
Viral Proteins ◽  
Post Inoculation ◽  
Farmed Fish ◽  
Asian Seabass ◽  
Cytopathic Effects ◽  
Transmission Electron ◽  
Acanthopagrus Latus ◽  
First Time

Scale drop disease virus (SDDV), an emerging piscine iridovirus prevalent in farmed Asian seabass Lates calcarifer in Southeast Asia, was firstly scientifically descripted in Singapore in 2015. Here, an SDDV isolate ZH-06/20 was isolated by inoculating filtered ascites from diseased juvenile yellowfin seabream into MFF-1 cell. Advanced cytopathic effects were observed 6 days post-inoculation. A transmission electron microscopy examination confirmed that numerous virion particles, about 140 nm in diameter, were observed in infected MFF-1 cell. ZH-06/20 was further purified and both whole genome and virion proteome were determined. The results showed that ZH-06/20 was composed of 131,122 bp with 135 putative viral proteins and 113 of them were further detected by virion proteome. Western blot analysis showed that no (or weak) cross-reaction was observed among several major viral proteins between ZH-06/20 and ISKNV-like megalocytivirus. An artificial challenge showed that ZH-06/20 could cause 100% death to juvenile yellowfin seabream. A typical sign was characterized by severe ascites, but not scale drop, which was considerably different from SDD syndrome in Asian seabass. Collectively, SDDV was confirmed, for the first time, as the causative agent of ascites diseases in farmed yellowfin seabream. Our study offers useful information to better understanding SDDV-associated diseases in farmed fish.

scholarly journals Identification, full-length genome sequencing, and field survey of citrus vein enation virus in Italy

2021 ◽  
Vol 60 (2) ◽  
pp. 293-301
Author(s):  
Maria MINUTOLO ◽  
Maria CINQUE ◽  
Giuseppe ALTAMURA ◽  
Francesco DI SERIO ◽  
Daniela ALIOTO ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Field Survey ◽  
Geographic Origin ◽  
Full Length ◽  
Full Genome Sequence ◽  
Post Inoculation ◽  
Campania Region ◽  
Italian Isolate ◽  
Full Length Genome ◽  
First Time

Citrus vein enation virus (CVEV) was described in Spain and then it has been reported in several citrus growing areas of Asia, America and Australia. Here, the occurrence of CVEV in Italy has been documented for the first time. The full genome sequence of a CVEV Italian isolate (14Q) was determined by high-throughput sequencing and the presence of the virus was confirmed by RT-PCR and graft-transmission to indicator plants, from which the virus was recovered six-months post-inoculation. Phylogenetic analysis based on the full-length genome of CVEV isolates from different countries showed that they are phylogenetically related to each other based on their geographic origin, rather than on their host and that the Italian isolate is more closely related to the Spanish isolate than to the other ones. A field survey revealed the presence of CVEV in some areas of Campania region (southern Italy), prevalently infecting lemon trees. In the frame of this survey, kumquat was identified for the first time as a host of CVEV. No symptoms were observed in the field so far. The infection of asymptomatic hosts and the transmission by aphid species present in Italy increase the risk that the virus could further spread.

scholarly journals An Agrobacterium mediated transformation system of guava (Psidium guajava L.) with endochitinase gene

2014 ◽  
Vol 14 (4) ◽  
pp. 232-237 ◽  
Author(s):  
Maneesh Mishra ◽  
Syed Uzma Jalil ◽  
Nimisha Sharma ◽  
Umesh Hudedamani
Keyword(s):  
Transformation Efficiency ◽  
Binary Vector ◽  
Nuclear Genome ◽  
Psidium Guajava ◽  
Nptii Gene ◽  
Cultivation Medium ◽  
Putative Transformants ◽  
Selection Medium ◽  
First Time

Genetic transformation of guava (Psidium guajava L.) was developed for the first time using in vitro grown shoot tip explant co-cultivated with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pIIHR-JBMch with endochitinase and nptII genes. The highest transformation efficiency was achieved by wounding explants with tungsten particles (0.5 µm) through particle acceleration system, followed by infection for 45 minutes with A. tumefaciens, grown overnight with 100 µM acetosyringone, corresponding to OD600=0.5 followed by co-cultivation for 72 hours under dark condition on co-cultivation medium (MS+100 µM acetosyringone+100 mg L-1 L-Cystein). Putative transformed explants regenerated shoots on selection medium stressed with 200 mg L-1 kanamycin for 12 weeks. Molecular analysis of putative transformants by PCR confirmed the integration of endochitinase and nptII gene in the plant nuclear genome.

Validation of chip grafting inoculation assay to assess the resistance of Solanum species against phytoplasma

2021 ◽  
pp. 1-5
Author(s):  
Khalid Pervaiz Akhtar ◽  
Najeeb Ullah ◽  
Muhammad Yussouf Saleem
Keyword(s):  
Solanum Species ◽  
New Delhi ◽  
Nested Polymerase Chain Reaction ◽  
Tomato Production ◽  
Disease Symptoms ◽  
Resistant Varieties ◽  
Polymerase Chain ◽  
Tomato Leaf Curl ◽  
First Time ◽  
Inoculation Assay

Abstract Big bud caused by several different phytoplasmas is an emerging threat to tomato production worldwide. The development of resistant varieties would be an effective approach to manage this problem, but it requires an appropriate screening technique. Recently, we have described a simple and efficient chip graft inoculation assay (CGIA) for the first time to screen tomato germplasm against Tomato leaf curl New Delhi virus. The present study was conducted to first validate the CGIA for phytoplasma transmission, then to assess the resistance of 74 genotypes belonging to different Solanum species against 16SrII-D phytoplasma. CGIA success rate and phytoplasma transmission was 100% since all the grafts survived and phytoplasma was detected in these plants using nested polymerase chain reaction. No genotype was found resistant as all the grafted plants showed typical disease symptoms. In addition to phytoplasma transmission, CGIA can be used for better understanding the plant–phytoplasma interactions and biology of phytoplasmas in tomato.

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